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| Status |
Public on Aug 05, 2010 |
| Title |
L2 polyA enriched 20dC 14hrs post-L1 N2 tiling array extraction2_array1 |
| Sample type |
RNA |
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| Source name |
L2 polyA enriched 20dC 14hrs post-L1 N2 tiling array extraction2_array1 channel_1
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
developmental stage: larva mid-L2 25dC 17.75 hrs post-L1
genotype: wild type
sex: Hermaphrodite
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| Growth protocol |
Animals were synchronized by treating a mixed stage hermaphrodite population with bleach to collect embryos. The embryos surviving bleach treatment were incubated without food, causing the larvae to arrest at the L1 stage upon hatching. Starved L1 larvae were plated in the presence of food and raised at 20/25 degrees Celsius (as specified).
The embryos were incubated without food, causing the larvae to arrest at the L1 stage upon hatching. Starved L1 larvae were plated on standard NGM plates in the presence of food (OP50) and raised at 25 degrees Celsius. Staging was determined by examining a subset of animals under Nomarski optics at intervals for vulval and germline development. Animals were collected at the mid-L2, mid-L3, mid-L4, and young adult (pre-gravid) stages and washed in S basal buffer. Exact times for growth after plating of starved L1s (all at 25 degrees C) were as follows: (1) L1 worms were grown for 4 hours (2) L2 worms were grown for 17.75 hours (Pn.p cells visible but not divided, gonad just starting to proliferate); (3) L3 for 26.75 hours (Pn.p cells divided once or twice; gonad just starting to turn up); (4) L4 for 34.25 hours (vulvae are in Christmas tree stage gonad has passed bend, sperm are present); and (5) Young Adult for 46 hours (vulvae fully formed and oocytes present in gonad, but no embryos). The worms were centrifuged on a sucrose cushion to remove debris and recovered in several washes of S basal.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For each library, a 10µg aliquot of DNaseI treated total RNA was separated into polyA+ and polyA- fractions using the MACSTM mRNA Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Double-stranded cDNAs were made from the polyA+ fractions using the Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen, Carlsbad, California, USA) and random hexamer primers (Invitrogen, 25 µM). The quality and quantity of the resulting double-stranded cDNAs was assessed using an Agilent DNA 1000 series II assay (Agilent Technologies, Santa Clara, CA, USA) and Nanodrop 7500 spectrophotometer (Nanodrop, Wilmington, DE, USA). Aliquots containing ~200 ng amplified cDNA were each diluted with water to 100uL and sonicated for 5 minutes. Sonication was performed in an ice water bath using a Sonic DismembratorTM 550 (Fisher, Ottawa, Ontario, Canada) with a power setting of "7" in pulses of 30 seconds interspersed with 30 seconds of cooling. Total sonication times refer to active sonication time only and do not include the rest periods in between each pulse.
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| Label |
biotin
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| Label protocol |
Array hybridization and processing performed at the Keck Facility at Yale University. Briefly, array hybridization buffer (100 mM MES, 1M [Na+], 20 mM EDTA, 0.01 % Tween 20) is used to prehybridize the array for 10-15 minutes at 450C. The prehybridized solution is removed and replaced with 80 ul of hybridization mixture containing hybridization buffer, fragmented cRNA (0.05ìg/ìl) and herring sperm DNA (0.1 mg/ml; Promega). Also included in the hybridization buffer are acetylated BSA (0.5 mg/ml, Invitrogen) and four control bacterial and phage cRNA (1.5 pM BioB, 5 pM Bio C, 25 pM BioD, and 100 pM Cre) samples to serve as internal controls for hybridization efficiency. The arrays are hybridized for 16 h at 450 in a rotisserie oven. After hybridization, arrays are washed using the Affymetrix fluidics station and stained with streptavidin-phycoerythrin (10 ìg/ml, Molecular Probes) according to the Affymetrix technical manual. Washed arrays will be scanned on a Affymetrix GeneChip Scanner 3000. Scanned output files will be visually inspected for hybridization artifacts and then analyzed by using Affymetrix GeneChip Operating Software (GCOS).
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| Hybridization protocol |
Array hybridization and processing performed at the Keck Facility at Yale University. Briefly, array hybridization buffer (100 mM MES, 1M [Na+], 20 mM EDTA, 0.01 % Tween 20) is used to prehybridize the array for 10-15 minutes at 450C. The prehybridized solution is removed and replaced with 80 ul of hybridization mixture containing hybridization buffer, fragmented cRNA (0.05ìg/ìl) and herring sperm DNA (0.1 mg/ml; Promega). Also included in the hybridization buffer are acetylated BSA (0.5 mg/ml, Invitrogen) and four control bacterial and phage cRNA (1.5 pM BioB, 5 pM Bio C, 25 pM BioD, and 100 pM Cre) samples to serve as internal controls for hybridization efficiency. The arrays are hybridized for 16 h at 450 in a rotisserie oven. After hybridization, arrays are washed using the Affymetrix fluidics station and stained with streptavidin-phycoerythrin (10 ìg/ml, Molecular Probes) according to the Affymetrix technical manual. Washed arrays will be scanned on a Affymetrix GeneChip Scanner 3000. Scanned output files will be visually inspected for hybridization artifacts and then analyzed by using Affymetrix GeneChip Operating Software (GCOS).
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| Scan protocol |
Array hybridization and processing performed at the Keck Facility at Yale University. Briefly, array hybridization buffer (100 mM MES, 1M [Na+], 20 mM EDTA, 0.01 % Tween 20) is used to prehybridize the array for 10-15 minutes at 450C. The prehybridized solution is removed and replaced with 80 ul of hybridization mixture containing hybridization buffer, fragmented cRNA (0.05ìg/ìl) and herring sperm DNA (0.1 mg/ml; Promega). Also included in the hybridization buffer are acetylated BSA (0.5 mg/ml, Invitrogen) and four control bacterial and phage cRNA (1.5 pM BioB, 5 pM Bio C, 25 pM BioD, and 100 pM Cre) samples to serve as internal controls for hybridization efficiency. The arrays are hybridized for 16 h at 450 in a rotisserie oven. After hybridization, arrays are washed using the Affymetrix fluidics station and stained with streptavidin-phycoerythrin (10 ìg/ml, Molecular Probes) according to the Affymetrix technical manual. Washed arrays will be scanned on a Affymetrix GeneChip Scanner 3000. Scanned output files will be visually inspected for hybridization artifacts and then analyzed by using Affymetrix GeneChip Operating Software (GCOS).
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| Data processing |
Tiling_Array_Signal_Extraction protocol. Mismatch (MM) probe values are subtracted from their corresponding perfect match (PM) probes. Processed data are obtained using following parameters: BPMap is BPmap:GPL5634:RW:1&oldid=22907 Tiling_Array_Normalization_and_Smoothing protocol. Biological replicates (usually 3 but sometimes 2) are normalized using quantile normalization. The normalized signal from multiple replicates are then smoothed to produce a composite signal. Each probe's value is replaced by the psuedomedian of all probes in a window around it from all replicates. This is done in a sliding window fashion shifted one probe at a time. The window size is set to 110bp, which corresponds to about 4 probes. Processed data are obtained using following parameters: window size is 110
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| Submission date |
Jul 28, 2010 |
| Last update date |
Aug 05, 2010 |
| Contact name |
DCC modENCODE |
| E-mail(s) |
[email protected]
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| Phone |
416-673-8579
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| Organization name |
Ontario Institute for Cancer Research
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| Lab |
modENCODE DCC
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| Street address |
MaRS Centre, South Tower, 101 College Street, Suite 800
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 0A3 |
| Country |
Canada |
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| Platform ID |
GPL5634 |
| Series (1) |
| GSE23266 |
mid-L2_polA_enriched_20dC_14hrs_post-L1_N2_tiling_array |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM571480_JYOU_CTIL_L2-2POA_041608.CEL.gz |
40.5 Mb |
(ftp)(http) |
CEL |
| Processed data are available on Series record |
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